Abstract
Purpose :
To develop new approach to control fungal keratitis (FK) by preparing nanobody (Nb) specific to β-glucan in fungal cell wall and synthesizing conjugate of Nb with natamycin (NAT).
Methods :
The expression recombinant vector of Nb was constructed by genetic engineering. Three sulfhydryl were introduced into the C-terminus of Nb. Nb and NAT are connected by covalent binding of functional groups. Antifungal activity of Nb and Nb-NAT against Aspergillus fumigatus was evaluated using MIC, PI uptake test, calcofluor white staining, aniline blue staining, biofilm biomass measurement, SEM and TEM. CCK-8 assay, ocular irritation and corneal fluorescein staining were utilized to detect the biocompatibility of Nb and Nb-NAT. In the eyes of mice, FK severity was evaluated by clinical score, plate counting and HE staining. The anti-inflammatory effect of Nb and NAT was examined by qRT-PCR and ELISA.
Results :
We found Nb specific to β-glucan could inhibit fungal growth by disrupting cell wall and biofilm formation of fungus. The content of β-glucan in fungus decreased by Nb treatment. Nb also could reduce the adhesion ability of fungal conidia to human corneal epithelial cells (HCECs). Further, we examined the capability of antifungal growth between NAT and Nb-NAT. Nb-NAT showed better antifungal effects than NAT. Moreover, Nb concentration below 0.5 mg/mL did not affect the viability of HCECs. Nb-NAT had less cytotoxicity and ocular surface irritation than NAT. Nb attenuated A.fumigatus virulence and relieve immune responses in FK. Nb-NAT treatment improved therapeutic effect compared with NAT. It decreased clinical scores and expression level of inflammatory factors.
Conclusions :
In this study, we prepared Nb specific to β-glucan and loaded the antifungal drug NAT along with Nb. Nb exerts significant antifungal and anti-inflammatory effects in FK. In addition, Nb-NAT exhibits prominent antifungal effect and had less cytotoxicity and corneal irritation compared with NAT. Nb-NAT treatment significantly alleviates corneal damage caused by fungal infection, providing a promising medical application to treat FK.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.